Bottom Line:
Quantitative mRNA and protein analysis corroborated the microarray results demonstrating enhanced expression of myogenic marker proteins and regulation of the expression of transcription factor GATA6 and its co-regulator, LMCD1.Finally, the pharmacological inhibition of endogenous S1P formation in response to TGFβ abrogated GATA6 up-regulation, supporting the view that the S1P pathway plays a physiological role in mediating the pro-myogenic effect of TGFβ.This study individuates GATA6 as novel player in the complex transcriptional regulation of mesoangioblast differentiation into SM cells and highlights a role for S1P to favour vascular regeneration.

ABSTRACTDifferent cells can contribute to repair following vascular injury by differentiating into smooth muscle (SM) cells; however the extracellular signals involved are presently poorly characterized. Mesoangioblasts are progenitor cells capable of differentiating into various mesoderm cell types including SM cells. In this study the biological action exerted by the pleiotropic sphingolipid sphingosine 1-phosphate (S1P) in human mesoangioblasts has been initially investigated by cDNA microarray analysis. Obtained data confirmed the anti-apoptotic action of this sphingolipid and identified for the first time a strong differentiating action toward SM cells. Quantitative mRNA and protein analysis corroborated the microarray results demonstrating enhanced expression of myogenic marker proteins and regulation of the expression of transcription factor GATA6 and its co-regulator, LMCD1. Importantly, GATA6 up-regulation induced by S1P was responsible for the enhanced expression of SM-specific contractile proteins. Moreover, by specific gene silencing experiments GATA6 was critical in the pro-differentiating activity of the cytokine TGFβ. Finally, the pharmacological inhibition of endogenous S1P formation in response to TGFβ abrogated GATA6 up-regulation, supporting the view that the S1P pathway plays a physiological role in mediating the pro-myogenic effect of TGFβ. This study individuates GATA6 as novel player in the complex transcriptional regulation of mesoangioblast differentiation into SM cells and highlights a role for S1P to favour vascular regeneration.

pone-0020389-g005: Role of GATA6 and LMCD1 transcription factors on the expression levels of SM markers induced by TGFβ in human mesoangioblasts (A) and their dependence on SphK activity (B,C).Cells transfected with scrambled-, GATA6-, LMCD1-siRNA A) and pre-treated with SKI-2 for 45 min (B,C) were stimulated with TGFβ and analyzed by Real-Time PCR.

Mentions:
In view of the here reported involvement of GATA6 and LMCD1 in the differentiating action of S1P, we next determined whether they were also involved in the mechanism by which TGFβ drives differentiation towards SM. As illustrated in Fig. 5A, GATA6 knock-down by specific RNA silencing strongly reduced the TGFβ-dependent enhancement of TPM1 mRNA and completely blocked that of the other key myogenic marker CNN1. Similarly, the down-regulation of LMCD1 mRNA abolished the TGFβ-induced increase of mRNA encoding for both SM markers TPM1 and CNN1. Interestingly, data presented in Fig. 5B show that TGFβ, similarly to S1P, was responsible for an increase of GATA6 mRNA content which was strongly reduced when SphK was pharmacologically inhibited. Likewise, SphK was involved in the increase of LMCD1 mRNA elicited by TGFβ (Fig. 5C). Thus, these results highlight a role for GATA6 and LMCD1 in the mechanism by which TGFβ exerts its pro-myogenic action.

pone-0020389-g005: Role of GATA6 and LMCD1 transcription factors on the expression levels of SM markers induced by TGFβ in human mesoangioblasts (A) and their dependence on SphK activity (B,C).Cells transfected with scrambled-, GATA6-, LMCD1-siRNA A) and pre-treated with SKI-2 for 45 min (B,C) were stimulated with TGFβ and analyzed by Real-Time PCR.

Mentions:
In view of the here reported involvement of GATA6 and LMCD1 in the differentiating action of S1P, we next determined whether they were also involved in the mechanism by which TGFβ drives differentiation towards SM. As illustrated in Fig. 5A, GATA6 knock-down by specific RNA silencing strongly reduced the TGFβ-dependent enhancement of TPM1 mRNA and completely blocked that of the other key myogenic marker CNN1. Similarly, the down-regulation of LMCD1 mRNA abolished the TGFβ-induced increase of mRNA encoding for both SM markers TPM1 and CNN1. Interestingly, data presented in Fig. 5B show that TGFβ, similarly to S1P, was responsible for an increase of GATA6 mRNA content which was strongly reduced when SphK was pharmacologically inhibited. Likewise, SphK was involved in the increase of LMCD1 mRNA elicited by TGFβ (Fig. 5C). Thus, these results highlight a role for GATA6 and LMCD1 in the mechanism by which TGFβ exerts its pro-myogenic action.

Bottom Line:
Quantitative mRNA and protein analysis corroborated the microarray results demonstrating enhanced expression of myogenic marker proteins and regulation of the expression of transcription factor GATA6 and its co-regulator, LMCD1.Finally, the pharmacological inhibition of endogenous S1P formation in response to TGFβ abrogated GATA6 up-regulation, supporting the view that the S1P pathway plays a physiological role in mediating the pro-myogenic effect of TGFβ.This study individuates GATA6 as novel player in the complex transcriptional regulation of mesoangioblast differentiation into SM cells and highlights a role for S1P to favour vascular regeneration.

ABSTRACTDifferent cells can contribute to repair following vascular injury by differentiating into smooth muscle (SM) cells; however the extracellular signals involved are presently poorly characterized. Mesoangioblasts are progenitor cells capable of differentiating into various mesoderm cell types including SM cells. In this study the biological action exerted by the pleiotropic sphingolipid sphingosine 1-phosphate (S1P) in human mesoangioblasts has been initially investigated by cDNA microarray analysis. Obtained data confirmed the anti-apoptotic action of this sphingolipid and identified for the first time a strong differentiating action toward SM cells. Quantitative mRNA and protein analysis corroborated the microarray results demonstrating enhanced expression of myogenic marker proteins and regulation of the expression of transcription factor GATA6 and its co-regulator, LMCD1. Importantly, GATA6 up-regulation induced by S1P was responsible for the enhanced expression of SM-specific contractile proteins. Moreover, by specific gene silencing experiments GATA6 was critical in the pro-differentiating activity of the cytokine TGFβ. Finally, the pharmacological inhibition of endogenous S1P formation in response to TGFβ abrogated GATA6 up-regulation, supporting the view that the S1P pathway plays a physiological role in mediating the pro-myogenic effect of TGFβ. This study individuates GATA6 as novel player in the complex transcriptional regulation of mesoangioblast differentiation into SM cells and highlights a role for S1P to favour vascular regeneration.